Diabetic nephropathy is a major cause of renal failure in the U.S. and develops in approximately 30% of insulin dependent diabetes mellitus (IDDM) patients. Recent studies by the Diabetes Control and Complications Trial Group have indicated that intensive insulin treatment substantially reduces the risk of developing complications, including nephropathy. However, the cost and effort of the intensive therapy, as well as the danger of hypoglycemic attacks dictate that this treatment should be limited to those patients who are prone to develop complications. It follows that an early selection of these diabetic subjects would be extremely helpful, but currently there are no adequate predictors available for clinical use.
Metabolic imbalance caused by hyperglycemia has been implicated as a major factor in the development of this condition and is associated with a genetic tendency to develop nephropathy. A prominent expansion of the mesangium with changes in the composition of the mesangial matrix have been observed in diabetic nephropathy (Williamson et al., Diabetes Met. Rev. 4:339 (1988), Steffes, M. W., et al. Diabetes 38:1077-81 (1989)).
Studies performed with human and experimental animal mesangial cells cultured in high-glucose medium have demonstrated an increased synthesis and accumulation of matrix proteins, namely collagens, including collagen type IV and fibronectin. This suggests that hyperglycemia plays a role in the mesangial changes of diabetic nephropathy. Ayo, S. H., et al. (1990a), Am. J. Pathol. 136:1339-1348; Nahman, N. S., et al., Kidney Int. 41:396-402 (1992); Danne, T., et al., Diabetes 42:170-177 (1993). The changes in the matrix secretion pattern of the cell are mediated either directly by hyperglycemia or by the glycation of mesangial matrix on prolonged exposure to high levels of glucose. Studies have demonstrated that cultured mesangial cells are influenced by the glycation of matrix leading to altered cell adhesion, spreading and proliferation. Since collagen IV (cIV) is the major component of the mesangial matrix (about 60%), changes in the interactions between this major mesangial glycoprotein and mesangial cells may play an important role in the pathology of diabetic nephropathy. Kim, Y., et al., Am. J. Pathol. 138:413-420 (1991). The changes in matrix deposition are secondary in time to insulin insufficiency. Altered matrix deposition including basement membrane thickening is also found in a variety of arterioles and arteries in patients with diabetes mellitus. Altered matrix deposition is found in the pancreas of diabetic patients. Altered matrix deposition puts diabetic patients at risk for developing secondary pathological changes including, but not limited to nephropathy, myocardial infarction, cerebral stroke, problems associated with reduced circulation, retinopathy, neuropathies and the like.
Cell-matrix interactions are mediated, for the most par; by a family of receptors known as integrins. The very late antigen (VLA) subgroup of integrins which share a common xcex21 chain, include the cell membrane receptors for cIV, xcex11xcex21 and xcex12xcex21. Although integrins are mainly studied for their role in cell differentiation, migration and signaling events, they may also be involved in the maintenance of tissue structure. For instance, cells can modify their matrix by altering the production of matrix proteins and/or by regulating matrix organization. Cells cultured under high glucose conditions resulted in an increased production of matrix components by mesangial cells. (Kashgarian, M., et al., Kidney Int. 41:524-529 (1992).) The balance of cell surface integrin expression has been demonstrated to be altered in various disease states including inflammation and malignancy (Waes and Carey, Otolarnyngologic Clinics of North America 25(5):1117 (1992); Adams, J. C., et al., Cell 63:425-435 (1990); Rozzo et al., FEBS Letters 332:263 (1993)). This altered expression has been associated with altered adhesion to extracellular components.
Presently, the only earliest available indicator of kidney changes is microalbuminuria which occurs after the appearance of nephropathic changes. Yet only a percentage of individuals with microalbuminuria go on to develop glomerulopathy. Individuals at risk for developing glomerulopathy are best treated with intense glucose-modulating therapies that have their own risk. Often physicians are hesitant to place individuals with microalbuminuria on such therapies since the majority of these patients do not proceed to glomerulopathy. Biopsies indicating the accumulation of matrix accompanying the expansion of the mesangium occur at a point when the process has become irreversible. Therefore an early predictor of nephropathy or other disease states associated with altered matrix deposition would be beneficial as an indicator of those patients who require stringent control of blood glucose levels to minimize nephropathic and other altered matrix deposition-associated disorders.
Thus, there is a need to identify markers associated with the changes seen in nephropathy and in other altered matrix deposition-associated disorders for the diagnosis of these disorders. There is a need to identify changes in regulation and function of integrins in diabetic patients and there is a need to develop a diagnostic test that can be used to identify patients who are likely to develop or have the early symptoms of nephropathy.
Alterations in the amounts and patterns of alpha-integrin subunits has now been correlated to the onset of nephropathy. Analysis of alpha integrin subunit expression as compared with controls provides a diagnostic tool for the determination of patients likely to develop severe nephropathy and a method to monitor progress of disease during treatment protocols.
Cells that express alpha integrins, such as kidney tissue, fibroblasts, endothelial cells, and blood cells are analyzed for alpha integrin subunit expression, for example, by in situ hybridization methods. Changes in the amounts and pattern of integrin subunit expression as compared with control samples, is diagnostic of nephropathy and can be used to screen individuals, e.g., diabetic patients at risk for developing severe disease.
Analysis of xcex11, xcex12, xcex13, xcex15, and beta-1 integrin subunit expression as compared with control tissue expression is preferred. An increase in xcex12, xcex13, xcex15, or beta-1 integrin expression and/or a decrease in xcex11 expression is diagnostic of increased risk of nephropathy. An especially preferred diagnostic method is the comparison of xcex11 and xcex12 integrin subunit expression with control tissue. A pattern change including a decrease in xcex11 and an increase in xcex12 is diagnostic of increased risk of nephropathy or onset of the disease.